The ubiquitous tripeptide L-glutathione (GSH) (gamma-glutamyl-cysteinyl-glycine), is a well-known biological antioxidant, and in fact is believed to be the primary intracellular antioxidant for higher organisms. When oxidized, it forms a dimer (GSSG), which may be recycled in organs having glutathione reductase. Glutathione may be transported through membranes by the sodium-dependent glutamate pump. Tanuguchi, N., et al. Eds., Glutathione Centennial, Academic Press, New York (1989). Glutathione is widely distributed in Nature, including yeast cells, botanic life and animals. It is made in the same way in humans by two different enzymes, and this is relevant to understanding the properties of glutathione (12).
GSH is “bent” in the same way across thousands of life forms, by two enzymes, and has evolved over a long time to specifically position its critical thiol. For comparison, the thiol of cysteine, alone, is overexposed and over reactive, and the thiol of homocysteine even more so, creating a highly destructive sulfur radical that disrupts endothelium in homocysteinemia. N-acetyl cysteine (NAC), when taken orally, loses the N-acetyl in the stomach, thereby leading to uncontrolled oxidation and a range of toxicities in humans.
The properties of GSH derive from its controlled reactivity, its ability to maintain a physiologically favorable Redox potential, its antioxidant properties in all subcellular compartments, the existence of avid glutathione transporters on cell membranes and mitochondria, and the fact that these properties are supported by enzymes: (i) that synthesize GSH; (ii) that amplify particular properties, such as GSH peroxidases and S-transferases and; (iii) enzymes that restore GSH after it has been used, GSH reductase. Loss of activity of glutathione peroxidase 4 (GPX4) in a process called ferroptosis, leads to accumulation of lipid-based reactive oxygen species (ROS), particularly lipid hydroperoxides.
GSH functions directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells from free-radical mediated damage. GSH is one of the primary cellular antioxidants responsible for maintaining the proper oxidation state within the body. GSH is synthesized by most cells, and is also supplied in the diet. GSH has been shown to recycle oxidized biomolecules back to their active, reduced forms. Because of the existing mechanisms for controlling interconversion of reduced and oxidized GSH, an alteration of the level of reduced GSH, e.g., by administration of GSH to an organism, will tend to shift the cells of the organism to a more reduced redox potential. Likewise, subjecting the organism to oxidative stress or free radicals will tend to shift the cells to a more oxidized potential. It is well known that certain cellular processes are responsive to redox potential. Reduced GSH produced in human adults from oxidized GSSG primarily by the liver, and to a smaller extent, by the skeletal muscle, red blood cells, and white cells. About 80% of the 8-10 grams glutathione produced daily is by the liver and distributed through the blood stream to the other tissues.
A deficiency of GSH in cells may lead to excess free radicals, which cause macromolecular breakdown, lipid peroxidation, buildup of toxins, and ultimately cell death. Because of the importance of GSH in preventing this cellular oxidation, GSH is continuously supplied to the tissues. However, under certain conditions, the normal, physiologic supplies of GSH are insufficient, distribution inadequate or local oxidative demands too high to prevent cellular oxidation. Under certain conditions, the production of and demand for GSH are mismatched, leading to insufficient levels on an organismal level. In other cases, certain tissues or biological processes consume GSH so that the intracellular levels are suppressed. In either case, by increasing the serum levels of GSH, increased amounts may be directed into the cells. In cellular uptake facilitated transport systems, the concentration gradient which drives uptake is increased.
As with all nutrients, eating or orally ingesting the nutrient would generally be considered a desired method for increase body levels thereof. Thus, attempts at oral GSH treatments were known. Prior work by the present inventor demonstrates efficacious administration by oral administration on an empty stomach. See, U.S. Pat. No. 6,159,500, and the below, each of which is expressly incorporated herein by reference. See also: U.S. Pat. Nos. 9,308,234; 9,062,086; 9,040,082; 8,911,724; 8,592,392; 8,361,512; 8,349,359; 8,252,325; 8,147,869; 8,114,913; 7,951,847; 7,709,460; RE40,849; 7,449,546; 7,449,451; 7,378,387; RE39,705; 7,078,064; 6,896,899; 6,835,811; 6,586,404; 6,423,687; 6,350,467; 6,262,019; 6,204,248; 6,197,749; 6,159,500; 9,265,808; 9,229,014; 9,149,451; 9,144,570; 8,981,139; 8,950,583; 8,734,316; 8,709,406; 8,679,530; 8,602,961; 8,591,876; 8,575,218; 8,518,869; 8,507,219; 8,501,700; 8,435,574; 8,426,368; 8,303,949; 8,221,805; 8,217,084; 8,217,006; 8,178,516; 8,093,207; 8,067,537; 7,923,045; 7,763,649; 7,723,327; 7,691,901; 7,615,535; 7,592,449; 7,579,026; 7,521,584; 7,407,986; 7,396,659; 7,384,655; 7,375,133; 7,371,411; 7,345,091; 7,320,997; 7,317,008; 7,279,301; 7,241,461; 7,238,814; 7,179,791; 7,169,412; 7,145,025; 7,094,550; 7,049,058; 7,045,292; 6,949,382; 6,896,899; 6,764,693; 6,709,835; 6,596,762; 6,586,404; 6,511,800; 6,444,221; 6,423,687; 6,395,494; 6,350,467; 6,346,547; 6,312,734; 6,262,079; 6,251,920; 6,204,248; 6,197,789; 6,166,090; 6,159,500; 6,069,167; 5,847,007; 5,770,609; 5,595,722; 5,545,569; 5,326,757; 5,204,114; 4,859,668; 20160082029; 2015028393; 2014027144; 2014019385; 2014014171; 20130317072; 20120244235; 20110151030; 20110129523; 20100291196; 20090311350; 20090042822; 20080234380; 20070065497; 20070053970; 20070026090; 20070004035; 20060105972; 20060008544; 20060008543; 20050226942; 20050222046; 20050090553; 20040105894; 20040071770; 20030211491; 20030129262; 20020136763; 20020002136; 20140256760; 20140045874; 20120245343; 20160158308; 20160068904; 20150246018; 20150209316; 20150038577; 20150030668; 20140271923; 20140271816; 20140100283; 20130202681; 20130129815; 20120244212; 20120141608; 20120135068; 20120087994; 20120021073; 20110305752; 20110111002; 20110077194; 20100316700; 20100291196; 20100233297; 20100233193; 20100166846; 20100166796; 20090176715; 20090068253; 20070053970; 20060099244; 20050239886; 20050222046; 20050130905; 20050123628; 20040157783; 20040022873; 20020182585; 20020136763; 20010000784. See, www.fda.gov/ucm/groups/fdagov-public/@fdagov-foods-gen/documents/document/ucm264131.pdf.
Metabolism of GSH.
The synthesis of GSH is dependent upon the availability of cysteine either supplied directly from the diet or cysteine or indirectly from methionine via the transsulfuration pathway. GSH synthesis and metabolism is governed by the enzymes of the γ-glutamyl cycle. GSH is synthesized intracellularly by the consecutive actions of γ-glutamylcysteinyl synthetase (Reaction 1) and GSH synthetase (Reaction 2). The action of the latter enzyme is feedback inhibited by GSH. The breakdown of GSH (and also of its oxidized form, GSSG) is catalyzed by γ-glutamyl transpeptidase, which catalyzes the transfer of the gamma-glutamyl moiety to acceptors such as SH-containing amino acids, certain dipeptides, and GSH itself (Reaction 3). The cellular turnover of GSH is associated with its transport, in the form of GSH, across cell membranes, where the majority of the transpeptidase is found. During this transport, GSH interacts with γ-glutamyl transferase (also known as transpeptidase) to form γ-glutamyl amino acids which are transported into cells. Intracellular γ-glutamyl amino acids are substrates of γ-glutamyl cyclotransferase (Reaction 4) which converts these compounds into the corresponding amino acids and 5-oxo-L-proline. The ATP-dependent conversion of 5-L-oxoproline to L-glutamate is catalyzed by the intracellular enzyme 5-oxo-prolinase (Reaction 5). The cysteinylglycine formed in the transpeptidase reaction is split by dipeptidase (Reaction 6). These six reactions constitute the γ-glutamyl cycle, which accounts for the synthesis and enzymatic degradation of GSH.
Two of the enzymes of the cycle also function in the metabolism of S-substituted GSH derivatives, which may be formed nonenzymatically by reaction of GSH with certain electrophilic compounds or by GSH S-transferases (Reaction 7). The γ-glutamyl moiety of such conjugates is removed by the action of γ-glutamyl transpeptidase (Reaction 3), a reaction facilitated by γ-glutamyl amino acid formation. The resulting S-substituted cysteinylglycines are cleaved by dipeptidase (Reaction 6A) to yield the corresponding S-substituted cysteines, which may undergo N-acetylation (Reaction 8) or an additional transpeptidation reaction to form the corresponding γ-glutamyl derivative (Reaction 3A).
Intracellular GSH is converted to its oxidized, dimeric form (GSSG) by selenium-containing GSH peroxidase, which catalyzes the reduction of H2O2 and other peroxides (Reaction 9). GSH is also converted to GSSG by transhydrogenation (Reaction 10). Reduction of GSSG to GSH is mediated by the widely-distributed enzyme GSSG reductase which uses NADPH (Reaction 11). Extracellular conversion of GSH to GSSG has also been reported; the overall reaction requires O2 and leads to the formation of H2O2 (Reaction 12). GSSG is also formed by reaction of GSH with free radicals. The GSH-dependent antioxidant system consists of GSH plus two enzymes: GSH peroxidase and GSH reductase. As this system operates, GSH cycles between its oxidized (GSSG) and reduced (GSH) forms.
Lipid hydroperoxides, which are formed during the peroxidation of lipids containing unsaturated fatty acids, are reduced, not by the usual GSH peroxidase, but by a special enzyme designed specifically to handle peroxidized fatty acids in phospholipids. This enzyme, known as phospholipid hydroperoxide GSH peroxidase is protein that can reduce both H2O2 and lipid hydroperoxides to the corresponding hydroxides (water and a lipid hydroxide, respectively). In contrast to the phospholipid hydroperoxide GSH peroxidase, ordinary GSH peroxidase is unable to act on lipid hydroperoxides.
Transport of GSH
The intracellular level of GSH in mammalian cells is in the range of 0.5-10 mM, while μM concentrations are typically found in blood plasma. Intracellular GSH is normally over 99% reduced form (GSH). The normal healthy adult human liver synthesizes 8-10 grams of GSH daily. Normally, there is an appreciable flow of GSH from liver into plasma. The major organs involved in the inter-organ transport of GSH are the liver and the kidney, which is the primary organ for clearance of circulating GSH. It has been estimated to account for 50-67% of net plasma GSH turnover. Several investigators have found that during a single pass through the kidney, 80% or more of the plasma GSH is extracted, greatly exceeding the amount which could be accounted for by glomerular filtration. While the filtered GSH is degraded stepwise by the action of the brush-border enzymes γ-glutamyltransferase and cysteinylglycine dipeptidase, the remainder of the GSH appears to be transported via an unrelated, Na+-dependent system present in basal-lateral membranes. GSH transported from hepatocytes interacts with the transpeptidase of ductile cells, and there is a substantial reabsorption of metabolites by ductule endothelium. In the rat, about 12 and 4 nmoles/g/min of GSH appear in the hepatic vein and bile, respectively.
GSH exists in plasma in four forms: reduced GSH (GSH), oxidized GSH (GSSG), mixed disulfide with cysteine (CySSG) and protein bound through a SH linkage (GSSPr). The distribution of GSH equivalents is significantly different than that of cyst(e)ine, and when either GSH or cysteine is added at physiological concentration, a rapid redistribution occurs. The distribution of GSH equivalents in rat plasma is 70.0% protein bound, with the remaining 30% apportioned as follows: 28.0% GSH, 9.5% GSSG, and 62.6% as the mixed disulfide with cysteine. The distribution of cysteine equivalents was found to be 23% protein bound, with the remaining 77% distributed as follows: 5.9% cysteine, 83.1% cystine, and 10.8% as the mixed disulfide with GSH. Plasma thiols and disulfides are not in equilibrium, but appear to be in a steady state maintained in part by transport of these compounds between tissues during the interorgan phase of their metabolism. The large amounts of protein-bound GSH and cysteine provide substantial buffering which must be considered in the analysis of transient changes in GSH and cysteine. This buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins. In erythrocytes, GSH has been implicated in reactions which maintain the native structure of hemoglobin and of enzymes and membrane proteins. GSH is present in erythrocytes at levels 1000 times greater than in plasma. It functions as the major small molecule antioxidant defense against toxic free radicals, an inevitable by-product of the erythrocytes' handling of 02.
GSH and the Immune System
Thiols and especially of GSH are important to lymphocyte function. Adequate concentrations of GSH are required for mixed lymphocyte reactions, T-cell proliferation, T- and B-cell differentiation, cytotoxic T-cell activity, and natural killer cell activity. Adequate GSH levels have been shown to be necessary for microtubule polymerization in neutrophils. Intraperitoneally administered GSH augments the activation of cytotoxic T-lymphocytes in mice, and dietary GSH was found to improve the splenic status of GSH in aging mice, and to enhance T-cell-mediated immune responses.
The presence of macrophages can cause a substantial increase of the intracellular GSH levels of activated lymphocytes in their vicinity. Macrophages consume cystine via a strong membrane transport system, and generate large amounts of cysteine which they release into the extracellular space. It has been demonstrated that macrophage GSH levels (and therefore cysteine equivalents) can be augmented by exogenous GSH. T-cells cannot produce their own cysteine, and it is required by T-cells as the rate-limiting precursor of GSH synthesis. The intracellular GSH level and the DNA synthesis activity in mitogenically-stimulated lymphocytes are strongly increased by exogenous cysteine, but not cystine. In T-cells, the membrane transport activity for cystine is ten-fold lower than that for cysteine. As a consequence, T-cells have a low baseline supply of cysteine, even under healthy physiological conditions. The cysteine supply function of the macrophages is an important part of the mechanism which enables T-cells to shift from a GSH-poor to a GSH-rich state.
The importance of the intracellular GSH concentration for the activation of T-cells is well established. It has been reported that GSH levels in T-cells rise after treatment with GSH; it is unclear whether this increase is due to uptake of the intact GSH or via extracellular breakdown, transport of breakdown products, and subsequent intracellular GSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cell activation in vitro. Depletion of intracellular GSH has been shown to inhibit the mitogenically-induced nuclear size transformation in the early phase of the response. Cysteine and GSH depletion also affects the function of activated T-cells, such as cycling T-cell clones and activated cytotoxic T-lymphocyte precursor cells in the late phase of the allogenic mixed lymphocyte culture. DNA synthesis and protein synthesis in IL-2 dependent T-cell clones, as well as the continued growth of preactivated CTL precursor cells and/or their functional differentiation into cytotoxic effector cells are strongly sensitive to GSH depletion.
The activation of physiologic activity of mouse cytotoxic T-lymphocytes in vivo was found to be augmented by intraperitoneal (i.p.) GSH in the late phase but not in the early phase of the response. The injection of GSH on the third day post immunization mediated a 5-fold augmentation of cytotoxic activity. Dietary GSH supplementation can reverse age-associated decline of immune response in rats, as demonstrated by maintenance of Concanavalin A stimulated proliferation of splenocytes in older rats.
GSH status is a major determinant of protection against oxidative injury. GSH acts on the one hand by reducing hydrogen peroxide and organic hydroperoxides in reactions catalyzed by GSH peroxidases, and on the other hand by conjugating with electrophilic xenobiotic intermediates capable of inducing oxidant stress. The epithelial cells of the renal tubule have a high concentration of GSH, no doubt because the kidneys function in toxin and waste elimination, and the epithelium of the renal tubule is exposed to a variety of toxic compounds. GSH, transported into cells from the extracellular medium, substantially protects isolated cells from intestine and lung are against t-butylhydroperoxide, menadione or paraquat-induced toxicity. Isolated kidney cells also transport GSH, which can supplement endogenous synthesis of GSH to protect against oxidant injury. Hepatic GSH content has also been reported to rise, indeed to double, in the presence of exogenous GSH. This may be due either to direct transport, as has been reported for intestinal and alveolar cells, or via extracellular degradation, transport, and intracellular resynthesis.
The nucleophilic sulfur atom of the cysteine moiety of GSH serves as a mechanism to protect cells from harmful effects induced by toxic electrophiles. The concept that GSH S-conjugate biosynthesis is an important mechanism of drug and chemical detoxification is well established. GSH conjugation of a substrate generally requires both GSH and GSH-S-transferase activity. The existence of multiple GSH-S-transferases with specific, but also overlapping, substrate specificities enables the enzyme system to handle a wide range of compounds.
Several classes of compounds are believed to be converted by GSH conjugate formation to toxic metabolites. Halogenated alkenes, hydroquinones, and quinones have been shown to form toxic metabolites via the formation of S-conjugates with GSH. The kidney is the main target organ for compounds metabolized by this pathway. Selective toxicity to the kidney is the result of the kidney's ability to accumulate intermediates formed by the processing of S-conjugates in the proximal tubular cells, and to bioactivate these intermediates to toxic metabolites.
The administration of morphine and related compounds to rats and mice results in a loss of up to approximately 50% of hepatic GSH. Morphine is known to be biotransformed into morphinone, a highly hepatotoxic compound, which is 9 times more toxic than morphine in mouse by subcutaneous injection, by morphine 6-dehydrogenase activity. Morphinone possesses an α,β-unsaturated ketone, which allows it to form a GSH S-conjugate. The formation of this conjugate correlates with loss of cellular GSH. This pathway represents the main detoxification process for morphine. GSH pretreatment protects against morphine-induced lethality in mouse.
The deleterious effects of methylmercury on mouse neuroblastoma cells are largely prevented by coadministration of GSH. GSH may complex with methylmercury, prevent its transport into the cell, and increase cellular antioxidant capabilities to prevent cell damage. Methylmercury is believed to exert its deleterious effects on cellular microtubules via oxidation of tubulin SH, and by alterations due to peroxidative injury. GSH also protects against poisoning by other heavy metals such as nickel and cadmium.
Because of its known role in renal detoxification and its low toxicity, GSH has been explored as an adjunct therapy for patients undergoing cancer chemotherapy with nephrotoxic agents such as cisplatin, in order to reduce systemic toxicity. In one study, GSH was administered intravenously to patients with advanced neoplastic disease, in two divided doses of 2,500 mg, shortly before and after doses of cyclophosphamide. GSH was well-tolerated and did not produce unexpected toxicity. The lack of bladder damage, including microscopic hematuria, supports the protective role of this compound. Other studies have shown that i.v. GSH coadministration with cisplatin and/or cyclophosphamide combination therapy, reduces associated nephrotoxicity, while not unduly interfering with the desired cytotoxic effect of these drugs.
GSH is relatively unstable in alkaline or oxidative environments, and is not absorbed by the stomach. It is believed that GSH is absorbed, after oral administration, if at all, in the latter half of the duodenum and the beginning of the jejunum. It was also believed that orally administered GSH would tend to be degraded in the stomach, and that it is particularly degraded under alkaline conditions by desulfurases and peptidases present in the duodenum. While GSH may be degraded, transported as amino acids, and resynthesized in the cell, there may also be circumstances where GSH is transported into cells without degradation; and in fact the administration of cysteine or cysteine precursors may interfere with this process.
Pure GSH forms a flaky powder that retains a static electrical charge, due to triboelectric effects, making processing and formulation difficult. The powder particles may also have an electrostatic polarization, which is akin to an electret. GSH is a strong reducing agent, so that autooxidation occurs in the presence of oxygen or other oxidizing agents. U.S. Pat. No. 5,204,114, provides a method of manufacturing GSH tablets and capsules by the use of crystalline ascorbic acid as an additive to reduce triboelectric effects which interfere with high speed equipment and maintaining GSH in a reduced state. The GSH is well absorbed, and distributed into the Peripheral Blood Mononuclear Cells (PBMC's) starting within 0.5 hours after oral ingestion. A certain crystalline ascorbic acid is, in turn, disclosed in U.S. Pat. No. 4,454,125. This crystalline form is useful as a lubricating agent for machinery. Ascorbic acid has the advantage that it is well tolerated, antioxidant, and reduces the net static charge on the GSH.
A number of disease states have been specifically associated with reductions in GSH levels. Depressed GSH levels, either locally in particular organs, or systemically, have been associated with a number of clinically defined diseases and disease states. These include HIV/AIDS, diabetes and macular degeneration, all of which progress because of excessive free radical reactions and insufficient GSH. Other chronic conditions may also be associated with GSH deficiency, including heart failure and coronary artery restenosis post angioplasty.
Clinical and pre-clinical studies have demonstrated the linkage between a range of free radical disorders and insufficient GSH levels. Diabetic complications may be the result of hyperglycemic episodes that promote glycation of cellular enzymes and thereby inactivate GSH synthetic pathways. GSH deficiency occurs diabetics, which explains the prevalence of cataracts, hypertension, occlusive atherosclerosis, and susceptibility to infections in these patients.
GSH functions as a detoxicant by forming GSH S-conjugates with carcinogenic electrophiles, preventing reaction with DNA, and chelation complexes with heavy metals such as nickel, lead, cadmium, mercury, vanadium, and manganese. GSH also plays a role in metabolism of various drugs, such as opiates. It has been used as an adjunct therapy to treatment with nephrotoxic chemotherapeutic agents such as cisplatin, and has been reported to prevent doxorubicin-induced cardiomyopathy. GSH is also an important factor in the detoxification of acetaminophen and ethanol, two powerful hepatotoxins.
Pharmacokinetics of GSH
The pharmacokinetics of intravenously administered GSH were determined in the rat and interpreted by means of an open, two-compartment model. Following a bolus injection of 50-300 mmol/kg GSH, arterial plasma concentrations of (i) GSH, (ii) oxidized GSH/GSSG, (iii) total thiols, and (iv) soluble thiols minus GSH, were elevated and then rapidly decreased non-exponentially, as anticipated. With increasing dose, the rate constant for drug elimination and plasma clearance increased form 0.84 to 2.44 mL/min. and the half-life of the elimination phase decreased from 52.4 to 11.4 minutes. Both the apparent volume of distribution and the degree of penetration of GSH into the tissues were diminished with increasing dose (from 3.78 to 1.33 L/Kg and from 6.0 to 0.51 as k12/k21, respectively). The data indicate that GSH is rapidly eliminated. This is mainly due to rapid oxidation in plasma rather than by increased tissue extraction or volume distribution. Thus, plasma GSH levels appear to be quickly regulated by which the body may maintain concentrations within narrow physiological limits.
When single doses of 600 mg GSH were administered intravenously to sheep, GSH levels in venous plasma and lung lymph rose transiently. The mean concentration was approximately 50 mM for venous plasma, peaking at 30 min, and returning to baseline after 45 minutes. Lung lymph peak level was about 100 mM at 15 min, returning to baseline after 30 minutes. Average epithelial lining fluid (ELF) levels were variable but showed no significant increase over baseline during the three hour observation period. Urine excretion was rapid with peak levels at 15 minutes. In both plasma and lung lymph, GSH accounted for greater than 95% of the total GSH (GSH plus GSSG). In ELF 75.4% of the baseline GSH was in the reduced form, whereas in urine 59.6% was present as GSH.
Orally ingested reduced GSH is absorbed intact from the small intestine in a rat model, specifically in the upper jejunum. It is noted that rat metabolism differs from man, and therefore the results of rat studies should be verified in man before the results are extrapolated. Plasma GSH concentration in rats increased from 15 to 30 mM after administration of GSH either as a liquid bolus (30 mM) or mixed (2.5-50 mg/g) in AIN-70 semi-synthetic diet (11). GSH concentration was maximal at 90-120 minutes after GSH administration and remained high for over 3 hours. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and re-synthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine (BSO) and acivicin showed that increased plasma GSH came mostly from absorption of intact GSH instead of GSH metabolites. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus, dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH.
Administration of oral GSH increased hepatic GSH levels in: (i) rats fasted 48 hours, (ii) mice treated with GSH depletors, and (iii) mice treated with paracetamol (a drug which promotes a depletion of hepatic GSH followed by hepatic centrilobular necrosis). In these experiments, the animals were orally intubated with 1000 mg/kg body weight GSH. Mean pretreatment values in 48-hour fasted rats were 3.0-3.1 mmol/g fresh hepatic tissue. Mean values after treatment were 5.8, 4.2, and 7.0 mmol/g fresh hepatic tissue for 2.5, 10, and 24 hours post-treatment, respectively. Mice were given an oral dose of GSH (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 hr in liver, kidney, heart, lung, brain, small intestine and skin. GSH concentrations in plasma increased from 30 mM to 75 mM within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. No increases over control values were obtained in most tissues except lung over the same time course. Mice pretreated with the GSH synthesis inhibitor BSO had substantially decreased tissue concentrations of GSH, and oral administration of GSH to these animals resulted in statistically-significant increases in the GSH concentrations of kidney, heart, lung, brain, small intestine and skin but not in liver.
The kinetics and the effect of GSH on plasma and urine SH were studied in ten healthy human volunteers. Following the intravenous infusion of 2000 mg/m2 of GSH the concentration of total GSH in plasma increased from 17.5±13.4 mmol/Liter (mean±SD) to 823±326 mmol/Liter. The volume of distribution of exogenous GSH was 176±107 Ml/Kg and the elimination rate constant was 0.063±0.027/minute, corresponding to a half-life of 14.1±9.2 minutes. Cysteine in plasma increased from 8.9±3.5 mmol/Liter to 114±45 mmol/Liter after the infusion. In spite of the increase in cysteine, the plasma concentration of total cyst(e)ine (i.e. cysteine, cystine, and mixed disulfides) decreased, suggesting an increased uptake of cysteine from plasma into cells. The urinary excretion of GSH and of cyst(e)ine was increased 300-fold and 10-fold respectively, in the 90 minutes following the infusion.
Normal healthy volunteers were given an oral dose of GSH to determine whether dietary GSH could raise plasma GSH levels. Results showed that an oral dose of GSH (15 mg/kg) raised plasma GSH levels in humans 1.5-10 fold over the basal concentration in four out of five subjects tested, with a mean value three times that of normal plasma GSH levels. Plasma GSH became maximal 1 hour after oral administration, dropping to approximately ½ maximal values after three hours. Equivalent amounts of GSH amino acid constituents failed to increase plasma levels of GSH. GSH bound to plasma proteins also increased with the same time course as seen with free GSH.